Efficient and versatile formation of glycosidic bonds via catalytic strain-release glycosylation with glycosyl ortho−2,2-dimethoxycarbonylcyclopropylbenzoate donors

Catalytic glycosylation is a vital transformation in synthetic carbohydrate chemistry due to its ability to expediate the large-scale oligosaccharide synthesis for glycobiology studies with the consumption of minimal amounts of promoters. Herein we introduce a facile and efficient catalytic glycosylation employing glycosyl ortho−2,2-dimethoxycarbonylcyclopropylbenzoates (CCBz) promoted by a readily accessible and non-toxic Sc(III) catalyst system. The glycosylation reaction involves a novel activation mode of glycosyl esters driven by the ring-strain release of an intramolecularly incorporated donor-acceptor cyclopropane (DAC). The versatile glycosyl CCBz donor enables highly efficient construction of O-, S-, and N-glycosidic bonds under mild conditions, as exemplified by the convenient preparation of the synthetically challenging chitooligosaccharide derivatives. Of note, a gram-scale synthesis of tetrasaccharide corresponding to Lipid IV with modifiable handles is achieved using the catalytic strain-release glycosylation. These attractive features promise this donor to be the prototype for developing next generation of catalytic glycosylation.


In the Supporting Information, synthetic procedures of the reactions in
26. In the Supporting Information, page S27, please correct the compound name of 3cb.
27. In the Supporting Information, section 3, please re-check the compound identifiers in the text and pictures.
Reviewer #3 (Remarks to the Author): The authors report a catalytic strain-release glycosylation employing glycosyl ortho-2,2dimethoxycarbonylcyclopropyl benzoates (CCBz). The new glycosylation method enabled the efficient synthesis of an array of glycosides. However, the preparation of the leaving group CCBzOH required several steps according to the reference, which renders the synthesis of glycosyl CCBz a daunting task. The authors claimed that various O-, S-, N-glycosidic bonds were efficiently constructed, but only one example of S-glycoside and only one example of N-glycoside were shown in Fig. 3. Based on the CCBz strategy, the formal synthesis of TMG-chitotrimycin, Nod factor, Myc factor, and lipid IV could be established, although they contain the similar skeletons. The mechanism of the new glycosylation was proposed. Further characterization of the key intermediates could be necessary to elucidate the mechanism. In the past few years, a series of catalytic glycosylation methods have been developed. Nevertheless, only several glycosylation methods more than 15 years ago were listed in the chronology of catalytic glycosylation of Fig.1. The description of the catalytic glycosylation has not fully reflected the recent development in this field. Overall, this reviewer feels that the manuscript is not suitable to publish in high-profile journals such as Nature Communications.
Reply: We thank the reviewer for pointing out these mistakes. The correct compound identifiers have been assigned to the corresponding compounds. For the corrected number series, please see revised How about the results if the glycosyl CCBz donor (the SePh leaving group in 14 was replaced by CCBz) was used?
Reply: Thank you for your keen question. We have indeed considered empoying a glycosyl CCBz donor to synthesize this tetrasaccharide, but we encountered some problems when synthesizing the donor. Specifically, the highly polar and acid-sensitive hemiacetal exhibited sluggish reactivity under normal coupling condition. However, in response to your and the second reviewer's interest in the synthesis of the tetrasaccharide using the glycosyl CCBz donor, we thus have revisited the synthesis of this donor and tried alternative synthesis approaches, and managed to prepare the N-2 Cbz protected glycosyl CCBz donor 15 by coupling the hemiacetal with in-situ generated CCBzCl (For experimental details, please refer to page S49 in revised supplementary information). When subjected to the strain-release glycosylation, the donor 15 gave corresponding tetrasaccharide in a slightly improved yield of 58% under the catalyzation of the Sc(III) catalyst. This result further demonstrates the effectiveness of the glycosyl CCBz donor in the synthesis of complex oligosaccharides.
To offer more useful information to the readers, sentences highlighted in yellow was added on page 11 in the revised manuscript, which read "We also prepared glycosyl CCBz donor 15 from glycosyl selenide 14 in two steps to test the viability of glycosyl CCBz in the tetrasaccharide assembly (For the experimental details, please see SI). To our delight, this glycosylation reaction gave an improved yield of 58% under established condition. The enhanced reactivity and convenient operation further demonstrate the usability of glycosyl CCBz." Again, we sincerely appreciate these valuable suggestions by the reviewer, which helped us to improve the quality of our paper significantly.
Response to reviewer 2: General Comment: Carbohydrate chemistry has been one of the most exciting aspects of organic chemistry in the last few decades. Many new glycosylation methods have come into being, however, the tedious optimizations and trial-and-error procedures are still required for the synthesis of structurally diversified glycans. Thus, more ideal glycosylation methods are demanded to improve the synthesis efficiency and to speed up the development of glycoscience. In this reviewer's opinion, an ideal glycosylation method should have following properties: 1) the donor can be easily manufactured and has good stability, 2) the donor can be efficiently activated under a mild condition, 3) the glycosylation can proceed in an orthogonal manner. This paper by Liu and co-workers reports an efficient catalytic glycosylation employing glycosyl ortho-2,2-dimethoxycarbonylcyclopropyl benzoates (CCBz) promoted by a readily accessible Sc(III) catalyst system. The versatile three carbon building blocks are useful in organic synthesis due to both their reactivity and ease of preparation. The Lewis acid catalyzed ring-opening of donor-acceptor cyclopropanes (DAC) using nucleophiles is one of the straightforward methods for rapid access to 1,3-bifunctional compounds. In this paper, a DAC structure was introduced into the glycosyl ester donor to construct a new glycosyl donor (CCBz) with new activation mode mediated by non-covalent interactions. This rationally designed new glycosyl donor with an intramolecularly incorporated DAC featuring a dual-functional anchor: the metallophilic 1,3-dicarbonyl group as the activation site, and the ensuing enolate as an acid scavenger. Various glycosyl CCBzs were synthesized from the corresponding hemiacetals by carbodiimide-mediated esterification in good-to-excellent yields. This glycosylation reaction performed well under the catalyzation of Sc(OTf)3 in various types of solvent. Acceptors ranged from aliphatic alcohols, sugar alcohols with primary, secondary and tertiary hydroxyl groups, benzoic acid derivative, phenol, sulfonamide and thiol were found to be compatible coupling partners in this glycosylation. A study on donor scope showed that glycosyl CCBzs derived from different monosaccharide-or oligosaccharide-based hemiacetals are competent coupling partners. Finally, this glycosylation method was applied successively to the synthesis of two chitooligosaccharide derivatives. Based on these results, this donor proved to be the prototype for developing next generation of catalytic glycosylation. The Supporting Information shows in detail the synthesis of compounds and documents the 1 H and 13 C NMR spectra of all new compounds. Accordingly, the reported method can be reproduced.
Overall, this reviewer thinks that this article is worthy of publication in Nature Communications due to the novelty and potential of this glycosylation method. However, there are still a lot of spaces for authors to improve the paper. Notably, many writing mistakes that exist in the manuscript and Supporting Information showed that more careful attention should be paid to the preparation of a high-quality research paper. Thus, major alterations are required before an acceptance in Nature Communications might be considered (see "Specific points" below).
Reply: We sincerely thank the reviewer's comments on our paper. Your valuable comments shed light on finding new directions to further improve our glycosyl donor and develop more interesting glycosylation reactions. We apologize for the typos and writing mistakes. After checking the manuscript again, the mistakes and typos, which are highlighted in red, have been corrected accordingly and listed here: 1) On page 1, the abstract part, "The salient features of our donor include the easy and scalable preparation of aglycon as well as the excellent thermostability, solubility and reactivity of the corresponding glycosyl CCBz in various organic solvent." has been corrected to "The salient features of our donor include the easy and scalable preparation of aglycon as well as the excellent thermostability, solubility and reactivity of the corresponding glycosyl CCBz in various organic solvents."; 2) On page 2, "glycosyl hetero-aromatic carboxylate esters" has been changed to "glycosyl heteroaromatic carboxylate esters".; 3) On page 6, "A series of Lewis acid were initially tested in the presence of 5 Å molecular sieve (MS) in a 0.05 M solution of 1,2-dichloroethane (DCE) at room temperature for 2 h." has been corrected to "A series of Lewis acids were initially tested in the presence of 5 Å molecular sieve (MS) in a 0.05 M solution of 1a in 1,2-dichloroethane (DCE) at room temperature for 2 to 5 h."; 4) On page 7, "revealing a orthogonal glycosyl ester type donors (entries 5-6)." has been corrected to "revealing a family of orthogonal glycosyl ester type donors (entries 5-6)."; 5) On page 8, "Our facile synthesis of 3m gave an example of catalytically feasible methods using cheap rare earth metal as the catalyst" has been corrected to "Our facile synthesis of 3m gave an example of the catalytically feasible method using cheap rare earth metal as the catalyst"; 6) On page 9, "L-rhamnosyl" has been corrected to "L-rhamnopyranosyl"; 7) On page 11, "The global deprotection of benzyl groups and benzyloxycarbonyl (CBz) group by hydrogenolysis in a mixed solvent of iPrOH/THF/H2O lead to the free tetrasaccharide 17 in 85% yield after purification." has been corrected to "The global deprotection of benzyl groups and benzyloxycarbonyl (CBz) group by hydrogenolysis in a mixed solvent of iPrOH/THF/H2O led to the free tetrasaccharide 17 in 85% yield after purification."; 8) On page 12, "The tetrasaccharides obtained has several orthogonally modifiable sites, which could be selectively furnished to obtain a series of Lipid IV derivatives to test their anti-bacterial activities." Has been corrected to "Several orthogonally modifiable sites on the tetrasaccharide could be selectively furnished to obtain a series of Lipid IV derivatives which hold the potential as bacterial TGases-targeting antimicrobial agents."; 9) Besides, the supplementary information was thoroughly checked, and the typos and wrong compound identifiers and compound names have been corrected.

Comment 1:
The title "Catalytic Strain-Release Glycosylation" is too simple to accurately reflect the emphasis and content of the paper.

Reply:
We thank the reviewer's valuable suggestion. After careful consideration, the prior title "Catalytic Strain-Release Glycosylation" has been amended to "Efficient and

Versatile Formation of Glycosidic Bonds via Catalytic Strain-Release Glycosylation with Glycosyl ortho-2,2-Dimethoxycarbonylcyclopropyl Benzoate Donors".
Comment 2: In the abstract, it's inappropriate to make the perspective like "With such, an array of peptidoglycan analogues could be prepared via the post-glycosylation modification strategy for novel antibiotic development to combat multidrug-resistant bacteria.". The discussion should focus on the developed method itself.
Reply: Thank you for your comment. The sentence has been removed in the revised manuscript.
Comment 3: In the figure 1, "a, the chronology of catalytic glycosylation" should contain more commonly used donors such as thioglycoside.

Reply:
We thank the reviewer's suggestion on Fig. 1. The purpose of Fig. 1a (2016)) catalyzed activation methods, the prevailing activation approaches of classical thioglycosides still require stoichiometric amount of thiophilic agents. Thus, we did not consider normal thioglycosides as meeting the categoria for our category of "catalytically activable glycosyl donors". Meanwhile, some modifications of thioglycosides on their anomeric leaving group have been reported that enable their catalytic activations (For CCBzOH could be obtained with over 95% purity by simply washing the resulting solid with hexane; (4) The utilization of CCBzOH as the efficient agent to achieve the activation of other unactivated hydroxyl group are still in progress in our lab. It is believed that this reagent will finally be accepted by the broad chemistry community for its versatility. We again thank the reviewer to raise the concern about the preparation of CCBzOH. To ensure that all of the reactions involved in the synthesis of CCBzOH can be reproduced by anyone who is interested in our glycosylation reactions, the detailed synthetic procedures for step-by-step decagram-scale preparation of CCBzOH with our tips and tricks are added in the supplementary information to ensure the reproducibility. For the modified section for the synthesis of CCBzOH, please see section 1, the synthesis of CCBzOH on page S7 in the revised supplementary information.

Comment 2:
The authors claimed that various O-, S-, N-glycosidic bonds were efficiently constructed, but only one example of S-glycoside and only one example of N-glycoside were shown in Fig. 3.

Reply:
We would like to thank this reviewer for the valuable comments, which have helped us to improve the manuscript. As per the reviewer's suggestion, we have removed the word "various" from the abstract to make our expression more precise. In order to further demonstrate the versatility of our glycosyl CCBz donor in S-glycosylation, two additional types of S-nucleophiles, aliphatic thiol 1-octanethiol 2r and sugar-derived thiol 2,3,4,6-tetra-O-benzoyl-1-thio-β-D-galactopyranose 2s were investigated. The coupling reaction with 2r proceeded efficiently to provide thioglycoside 3r in a yield of 64%. However, no reaction occurred when 2s was used as the acceptor. While this result was less satisfactory, it is believed that there is still a lot more for us to explore, and the reviewer's insightful suggestions have enlightened us about the new possibilities for our strain-release glycosylation. Specifically, we are now exploring the strain-release glycosylation of glycothiols, which promises access to the 1,1'-S-linked sugar derivatives of biological and medicinal interests. With respect to N-glycosylation, we are actively working on using the glycosyl CCBz donor to achieve the glycosylation reactions of other N-nucleophiles like nucleobases and asparagine derivatives. Since these N-nucleophiles usually have weaker nucleophilicity, the synthesis of N-glycosides is considered challenging. To further showcase the usefulness of glycosyl CCBz in the preparation of nucleosides and asparagine glycosides, we would like to publish these results in a separate paper. According to the reviewer's comment, additional lines highlighted in yellow have been added on page 9 in the revised manuscript, which read "Finally, the S-acceptors were also viable for the construction of S-linked glycosides, as exemplified by the efficient coupling reactions of donor 1a with aromatic thiol 2q and aliphatic thiol 2r, respectively. It should be noted that in all cases of successful glycosylation reactions employing heteroatom nucleophiles, the direct ring-opening of DAC moiety by acceptors was not observed, denoting the intramolecular cyclization can be kinetically favorable even in the presence of a heteroatom nucleophile. However, it was disappointing that when galactose-derived anomeric thiol 2s was subjected to our optimal condition, no reaction occurred. Currently, the reason for the unpleasant consequence was unknown and we are still working on the synthesis of 1,1'-thiosaccharide congeners by strain-release glycosylation because of their important roles as drug candidates. 65-67 ".
Comment 3: Based on the CCBz strategy, the formal synthesis of TMG-chitotrimycin, Nod factor, Myc factor, and Lipid IV could be established, although they contain the similar skeletons.
Reply: At first glance, the structures for these COS derivatives indeed appear similar; however, the strategies we deployed to synthesize chitooligosaccharides and Lipid IV tetrasaccharide vary in many aspects when testing the viability of glycosyl CCBz as glycosylating agents in the synthesis of complex oligosaccharides, each with its unique emphasis and purpose. For the chitooligosaccharide synthesis, an iterative glycosylation strategy was used to test the ability of strain-release glycosylation in a linear synthetic route. Meanwhile, because the TMG-chitotriomycin, Nod factor and Myc factor share the same tetrasaccharide skeleton except for the substitution patterns in the terminal monosaccharide, the main consideration was the protection fashion of the 2-amino group of the terminal glycosyl donor. In the initial manuscript, a selenoglycoside was used for the synthesis of tetrasaccharide, which might undercut the novelty of the work. However, by successfully synthesizing N-2 Cbz protected glycosyl CCBz donor and exposing it to the strain-release glycosylation, the challenging assembly of tetrasaccharide 16 was achieved in an improved yield of 58% under milder condition with cheap and abundant Sc(III) catalyst instead of a stoichiometric amount of promoter, demonstrating the usability of the glycosyl CCBz as an efficient glycosylating agent in the assembly of complex oligosaccharides. When it comes to the Lipid IV tetrasaccharide synthesis, a convergent strategy was selected to test the ability of oligosaccharide glycosyl CCBz donor for complex oligosaccharide synthesis. As has been demonstrated in the Fig. 6, the operation at the anomeric position and the glycosylation stage of disaccharide glycosyl CCBz are smooth to furnish the tetrasaccharide on a gram-scale. Meanwhile, because the peptidoglycan is not confined as the simple disaccharide or tetrasaccharide, the potential for the precise preparation of bigger oligomers of peptidoglycans was also considered by leaving an orthogonally cleavable TBS ether at the O-4 in the non-reducing terminal unit. Besides, in the overall synthesis, thioglycoside was used as the acceptor twice while the aglycon transfer side-reaction was almost suppressed. Considering the aglycon transfer is one of the most common side-reaction when the thioglycoside is involved in the glycosylation reaction as the acceptor (For one recent example cited in our paper, please see ref. 94, Xiao, G. Chem. Sci. 12, 5143-5151 (2021)), our method offered a more flexible choice of acceptor over other glycosylation reactions.